Structure and functional properties of large pore cellulose-based beads for chromatography of large biomolecules

The structure and functional properties of new large-pore, cellulose-based particles for biomolecule chromatography are evaluated. The particles are approximately 90 µm in diameter and contain interconnected pores with an average diameter of 3.6 µm and a macro-porosity of 0.76. Smaller pores, approximately 4 nm in diameter, are also present within the cellulose backbone. Because of the large macro-pore size and porosity, the particles have a high internal hydraulic permeability (0.08 μm²), similar to the permeability of typical monoliths. Thus, when these particles are packed in a column, a significant fraction of the mobile phase (about 0.018) flows within the particles themselves, resulting in a large convective enhancement of intraparticle transport rates. Experimental measurements with different proteins show flow rate-independent HETP for pulse injections of IgG, thyroglobulin, and IgM under non-binding conditions, residence time-independent dynamic binding capacity for frontal loading of IgG and IgM in the range 1-0.25 min, and constant peak width for gradient elution of IgG at residence times between 2 and 0.25 min. The results are consistent with a perfusion chromatography model, which accurately predicts frontal loading and gradient elution results using parameters obtained from the non-binding HETP data. The model can be used to assess performance when using these materials for different biomolecules and bioparticles. Compared to existing resins, dynamic binding capacities of the new resin are smaller for smaller proteins and at long residence times, but comparable to those obtained with monoliths for larger proteins and short residence times.